Foamy virus mutant reverse transcriptase

ABSTRACT

The present invention relates to a highly processive reverse transcriptase having DNA polymerase activity and substantially reduced protease activity. More specifically, the invention relates to an isolated reverse transcriptase from foamy virus comprising a substantially inactivated protease. The invention also relates to vectors containing the gene and hosts transformed with the vector of the invention. Further, the invention relates to a method for producing reverse transcriptase having DNA polymerase activity and substantially reduced protease activity by expressing the reverse transcriptase genes of the present invention in a recombinant host. Methods are also provided for producing cDNA from polynucleotides using the highly processive reverse transcriptase of the invention. Kits for the preparation of cDNA from RNA comprising the highly processive reverse transcriptase of the invention are also provided.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 United States National phaseapplication of PCT/US02/16528, filed May 22, 2002, which claims priorityto United States Patent Application Ser. No. 60/292,994, filed May 22,2001, incorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported by a grant from the National Cancer Institute(No. CA18282 and CA09229)

BACKGROUND OF THE INVENTION

Foamy Viruses (FVs; Spumavirinae) are classified as retroviruses anddemonstrate classic retroviral genomic organization, including the threehallmark genes gag, pol, and env (FIG. 1; Rethwiln, Curr. Top.Microbiol. Immunol. 193:1-24 (1995)). Despite this classification andorganization, several aspects of the FV life cycle differ significantlyfrom retroviruses. Three of these differences highlight uniqueproperties of the polymerase (Pol) protein and the reverse transcriptase(RT) enzyme encoded within it. First, FV has an unusual mechanism forthe expression of Pol. Typical retroviruses express Pol as part of aGag-Pol fusion protein, which mediates Pol incorporation into the virionthrough Gag-Gag interactions. In contrast, FV Pol is expressed from itsown spliced message, and consequently FV must employ a unique strategyfor incorporation of Pol into the viral particle (Lochelt and Flugel, J.Virol. 70:1033-1040 (1996), Yu et al. Science 271:1579-1582 (1996)).Second, reverse transcription occurs at a different stage in the FV lifecycle. Unlike conventional retroviruses, FV particles contain DNA thatappears to be used as the functional genome when infecting a new cell(Moebes et al., J. Virol. 71:7305-7311 (1997), Yu et al., J. Virol.73:1565-1572 (1999)). This requires that FV RT be active during, orshortly after particles bud from an infected cell. Third, the FV Polpolyprotein undergoes limited processing. A single cleavage eventbetween RT and Integrase (IN) takes place resulting in two matureenzymatic proteins, IN and a PR-RT fusion protein (Pfrepper et al.,Virus Genes 22:61-72 (2001)).

Because of its central role in the retroviral life cycle, RT has been atarget of drugs designed to inhibit HIV-1 replication and controlinfections. One major class of these RT inhibitors is the nucleosideanalog inhibitors, or chain terminators, which include (AZT) and (3TC).In vitro, these drugs have proven to be potent inhibitors of RT activityand viral replication for many retroviruses, including HIV-1. However,it has been demonstrated previously that of 3TC, AZT, and ddI, only AZTspecifically inhibits SFVcpz (hu), (a human foamy virus newly designatedPrototype Foamy Virus (PFV)), replication (Yu et al., J. Virol.73:1565-1572 (1999)).

In HIV-1 patients, these drugs have been shown to inhibit viralreplication, but HIV-1 is able to rapidly evolve mutations that allow itto overcome this inhibition. In the case of 3TC, resistance rapidly andconsistently develops through mutation of the second residue in theHIV-1 catalytic motif Tyr Met Asp Asp (YMDD) to valine (Tisdale et al.Proc. Natl. Acad. Sci. USA 90:5653-5656 (1996)). The YXDD (SEQ ID NO:17) catalytic motif, wherein X defines any amino acid residue, is highlyconserved among all reverse transcriptases, and all retroviral RTscontain a methionine in the second residue with the exception of MurineLeukemia Viruses (MLVs) and FVs, which contain a valine in thisposition. When the amino acid sequence of SFVcpz(hu) RT (PFV RT) iscompared to other retroviral RTs, the degrees of homology range fromabout 27% (HIV-2) to about 34% (Mo-MLV) (Maurer et al. J. Virol.62:1590-1597 (1988)). Despite this low level of overall homology, PFV RTcontains residues shown to be functionally essential in all otherretroviral RTs, including the YXDD motif (SEQ ID NO: 17). Interestingly,both MLV and FV contain a valine in their catalytic YXDD motifs (SEQ IDNO: 17), and both are naturally resistant to 3TC.

In the context of the virus, RT is expressed as part of a larger Polpolyprotein, which also contains Integrase (INT) and Protease (PR).Unlike other retroviruses, the FV Pol undergoes a single cleavage eventto release INT, leaving a mature PR-RT fusion protein. However, inbacterial over-expression systems a second cleavage event between PR andRT has been previously observed. To avoid this artificial cleavage, asprovided herein the entire PR-RT fusion protein was bacteriallyexpressed with a point mutation in the PR active site termed D/A,wherein an alanine replaces an aspartic acid residue. Both wild type anda mutated (V313M) RTs were successfully expressed and purified in theD/A context.

Further, it was determined from mutations in the RT which reduce theactivity of the RT by about 45 to 60% that foamy virus (FV) productionwas inhibited or stopped when RT activity was reduced. Therefore, unlikeother retroviruses FV requires a highly active reverse transcriptase. Ithas also been determined by the present invention that FV requires areverse transcriptase that is highly processive, i.e., capable ofproducing long nucleotide transcription products. In one embodiment ofthe present invention an isolated FV RT has been found to be capable ofgenerating products that were well above 600 base pairs(bp) in 10minutes. In similar studies the processivity of HIV-1 reversetranscriptase has been measured to generate transcript products of 150bp or smaller (Boyer and Hughes, Microbiol. Agents Chemother. 39:1624-8(1995)).

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an isolated foamy virusprotease-reverse transcriptase polyprotein having highly processivereverse transcriptase activity and substantially reduced proteaseactivity. In particular, the invention relates to polyproteins whereinthe nucleic acid which encodes the foamy virus protease-reversetranscriptase has been mutated so as to functionally inactivate theprotease activity. In one particular embodiment the nucleic acidsequence that encodes the aspartic acid in the catalytic site of theprotease is changed, for example, to encode alanine.

The invention also provides for the production of the isolated foamyvirus protease-reverse transcriptase having DNA polymerase activity andsubstantially reduced protease activity. Vectors and plasmids whichcomprise nucleic acids which encode the foamy virus protease-reversetranscriptase polyproteins and recombinant host cells are alsodisclosed. Kits for the production of cDNA from polynucleotides, e.g.,RNA and the like, comprising containers having a foamy virus proteasereverse transcriptase of the present application, a primer, nucleotidetriphosphates and the necessary buffers comprise another embodiment ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the PFV genome and encodedproteins. Pfv is a complex retrovirus encoding six open reading frames(gray boxes). The Pol protein is synthesized independently of Gag andcontains the enzymes protease, reverse transcriptase, and integrase. Thearrow indicates the single Pol cleavage site.

FIG. 2 depicts the virion-associated RT activity of PFV RT-V313M on ahomopolymeric template. FAB cells were transfected with viral orirrelevant (mock) plasmid DNA, extracellular virions were harvested 4days post-transfection and concentrated. RT activity was determined foreach of the concentrated samples by measuring incorporation of aradiolabeled nucleotide on a poly (A);d(T) template primer over time.Each time point was normalized to the mode sample.

FIG. 3 depicts a diagram of NcoI sites in the viral plasmid used fortransfection and predicted sizes of resulting products that hybridize tothe probe. The shaded gray regions of the DNA indicate the location ofthe LTRs. The asterisk indicates the size of the fragment correspondingto the unique cDNA fragment.

FIG. 4 depicts the 3TCTP inhibition curves for HIV-1, FV D/A, and FVD/A-RTVM recombinant RTs. Polymerization using a heteropolymerictemplate by purified recombinant RTs was measured by incorporation ofradiolabeled dCTP in the absence or presence of 3TCTP at concentrationsof 0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM. HIV-1 RT (♦), FV D/A-RT (▪), and FVD/A-RTVM (▴) were tested.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to the production of an isolated highlyactive and highly processive foamy virus reverse transcriptase havingDNA polymerase activity and substantially reduced protease activity. Inparticular, the isolated reverse transcriptase is a modified foamy virusprotease-reverse transcriptase polyprotein having a functionallyinactivated protease. Recombinant plasmids and vectors of the presentinvention provide for the production of the highly processive isolatedreverse transcriptase for use in molecular and recombinant DNA methods,such as for the in vitro synthesis of cDNA from RNA, i.e., mRNA, toreplace less processive enzymes used currently.

By the term “substantially no protease activity” is intended a purifiedor isolated foamy virus protease-reverse transcriptase (PR-RT)polyprotein or fusion protein having a functionally inactivated proteasewhich does not cleave the foamy virus PR-RT fusion protein whenoverexpressed in a bacterial host cell.

The term “functionally inactivated” is intended to define pointmutations and small amino acid deletions or insertions, i.e., up to 10amino acid residues, in the protease of the foamy virus PR-RT fusionprotein such that the protein retains high reverse transcriptaseactivity and has substantially reduced protease activity.

The foamy virus, or spumaviriae virus, reserve transcriptase cancomprise a number of types, including for example, that isolated fromnonhuman primates (Hooks and Detrick-Hooks, in Kurstak and Kurstak(eds.), Comparative Diagnosis of Viral Disease, Vol. 4, Academic Press,Inc., New York (1981), cows (Malanquist et al., Cancer Res. 16:188-200(1969), cats (Fabricant et al., Cornell Vet. 59:667-672 (1969); hamsters(Hruska and Takemoto, J. Natl. Cancer Inst. 54:601-605(1975), and humans(Achong et al., J. Natl. Cancer Inst. 42:299-307 (1971), Mauer et al.,J. Virol. 62:1590-1597 (1988)).

Reverse transcriptase (RT) of foamy virus appears to act as part of aprotease-reverse transcriptase polyprotein or fusion protein in infectedcells. Further, data indicates that the presence of the protease, or aportion thereof, contributes to the active structure of the reversetranscriptase. Unlike other retroviruses, FV Pol is expressed from isown spliced message. Although the mechanism for viral packaging the Polmust interact specifically with either the Gag protein, the genomic RNA,or both to ensure encapsidation.

PR-RT proviral DNA can be isolated using standard isolation techniques.The DNA can be cleaved or fragmented into linear segments. Fragmentationcan be accomplished by, for example, use of enzymes which digest orcleave DNA, i.e., restriction enzymes, or mechanical forces. Afterfragmentation of the DNA, the segments are separated by standardmethods. Description of such recombinant DNA methods can be found in,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Sambrook and Russell, Molecular cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001), each incorporated herein by reference.

The DNA fragments encoding the foamy virus PR-RT gene can beaccomplished in any number of ways well known to the skilled artisan.For example, the DNA fragments can be referenced (Maxam and Gilbert,Meth. Enzymol. 64:499 (1980); Messing, Meth. Enzymol. 101C:20 (1983)).Alternatively, hybridization can be employed using a labeled, i.e., aradioactive, fluorescent, or luminescent label, DNA probe (Southern, J.Mol. Biol. 98: 503 (1975); Benton and Davis, Science 196:180 (1977);Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72: 3961-65 (1975)).

The identified fragments can be pooled, ligated into a suitable vector,used to transform or transfect a host cell. Transformed host cells arescreened for production of PR and RT activity using methods well knownto the skilled artisan as described herein. Alternatively, clones oftransformed host cells can be screened and identified by hybridizationwith complimentary labeled oligonucleotide probes specific for theprotease and reverse transcriptase gene.

In another embodiment, PR-RT nucleotide sequences further includederivatives (e.g., nucleotide sequence variants), such as those encodingother possible codon choices for the same amino acid or conservativesubstitutions thereof, such as, for example, naturally occurring allelicvariants. Due to degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as afoamy virus PR-RT can be used in the present invention. These include,but are not limited to, nucleotide sequences comprising substantiallyall or a portion of the gene encoding foamy virus PR-RT fusion proteinhaving reverse transcriptase activity which is altered by thesubstitution of different codons that encode the same or a functionallyequivalent amino acid residue (e.g. a conservative substitution) withinthe sequence, producing a silent change.

Foamy virus PR-RT nucleic acids further include those nucleic acidshybridizable to, or complementary to, the SFVcpz(hu) sequence(Herchenroder et al., Virology 201:187-199 (1994), incorporated hereinby reference in its entirety). This isolate has been recently designatedPrototype Foamy Virus (PFV) and this designation will be used throughoutthe present disclosure. In a specific embodiment, a nucleic acid that ishybridizable to a foamy virus PR-RT nucleic acid, or to a nucleic acidencoding a foamy virus PR-RT derivative, under conditions of low, mediumor high stringency is provided. Low, moderate, and high stringencyconditions are well known to those of skill in the art, and will varypredictably depending on the base composition of the particular nucleicacid sequence and on the specific organism from which the nucleic acidis derived. For guidance regarding such conditions see, for example,Sambrook et al., (1989) supra, incorporated herein by reference.

An alternative to isolating the PR-RT gene from a foamy virus proviralDNA is to synthesize the nucleic acid by standard methods well known inthe art (e.g., by use of a commercially available automated DNAsynthesizer).

In another embodiment, polymerase chain reaction (PCR) can be used toamplify the desired sequence in a genomic or cDNA library, prior toselection. Oligonucleotide primers representing known foamy virus PR-RTsequences can be used as primers in PCR. The synthetic oligonucleotidecan be utilized as primers to amplify particular oligonucleotides withinthe FV PR-RT gene by PCR sequences from a source (RNA or DNA), typicallya cDNA library, of potential interest. PCR can be carried out, forexample, by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp). The DNA being amplified can include mRNA or cDNAor genomic DNA.

The identified and isolated FV PR-RT nucleic acids encoding a PR-RTpolyprotein having substantially reduced protease activity can then beinserted into an appropriate cloning vector. A large number ofvector-host systems known in the art can be used. Possible vectorsinclude, but are not limited to, plasmids or modified viruses. Thevector system is selected to be compatible with the host cell. Suchvectors include, but are not limited to, bacteriophages such as lambdaderivatives, yeast integrative and centromeric vectors, 2μ plasmid, andderivatives thereof, or plasmids such as pT5m, pBR322, pUC, pcDNA 3.1 orpRSET (Invitrogen) plasmid derivatives or the Bluescript vector(Stratagene) to name a few. The insertion of the FV PR-RT nucleic acidof the present invention into a cloning vector can, for example, beaccomplished by ligating the nucleic acid, e.g. DNA, fragment into acloning vector that has complementary cohesive termini. If thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, however, the ends of the DNA molecules can beenzymatically modified. Alternatively any desired restrictionendonuclease site can be produced by ligating nucleotide sequences(e.g., linkers) onto the DNA termini, these ligated sequences cancomprise specific chemically synthesized oligonucleotides encodingrestriction endonuclease recognition sequences. In an alternativemethod, the cleaved vector and FV PR-RT nucleic acids can be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells by, for example, transformation, transfection, infection,electroporation, and the like, so that many copies of the nucleic acidsequence are generated.

The nucleic acid encoding FV PR-RT of the present invention or afunctionally active derivative thereof can be inserted into anappropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedpolypeptide coding sequence. The necessary transcriptional andtranslational signals can also be supplied by the native FV PR-RT geneand/or its flanking regions. A variety of host-vector systems can beutilized to express the FV PR-RT polyprotein or fusion protein. Theseinclude, but are not limited to, mammalian cell systems infected withvirus, (e.g., vaccinia, adenovirus, modified foamy virus, and the like),insect cell systems infected with virus, e.g., baculovirus,microorganisms such as yeast, or transformed bacteria. The expressionelements of vectors vary in their strength and specification. Dependingon the host-vector system utilized, any one of a number of suitabletranscription and translation elements can be used. In a specificembodiment, the PFV PR-RT of the present invention is expressed, or anucleic acid sequence encoding a functionally active reversetranscriptase and a functional inactive protease is expressed inmammalian cells or bacteria.

Any of the methods previously described for the insertion ofoligonucleic acid (ONA) fragments into a vector can be used to constructexpression vectors containing a chimeric gene consisting of appropriatetranscriptional translational control signals and the polyprotein codingsequences. These methods include in vitro recombinant DNA and syntheticmethods and in vitro recombinants (genetic recombination). Expression ofnucleic acid sequences encoding the FV PR-RT fusion protein of thepresent invention can be regulated by a second nucleic acid sequence sothat the FV PR-RT of the invention is expressed in a host transformedwith the recombinant DNA molecule. For example, expression of a FV PR-RTpolyprotein can be controlled by any promoter/enhancer element known inthe art. Promoters that can be used include, but are not limited to, theSV40 early promoter region (Benoint and Chambon, Nature290:304-310(1981)), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)),the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.Sci. USA 78:1441-1445(1981)), the regulatory sequences of themetallothionein gene (Brinster et al., Nature 296:3942 (1982)),prokaryotic expression promoters such as β-lactamase promoter(Villa-Komanoff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978))or the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25(1983)), just to name a few.

Expression vectors containing FV PR-RT nucleic acid inserts of thepresent invention can be identified by general approaches well know tothe skilled artisan, including: (a) nucleic acid hybridization, (b) thepresence or absence of “marker” gene function, and (c) expression of theencoded FV PR-RT fusion protein having a substantially functionallyinactive protease and an active reverse transcriptase. Once a particularrecombinant DNA molecule is identified and isolated, several methodswell known to the skilled artisan can be used for propagation. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity.

In order to prepare a nucleic acid that encodes FV PR-RT having highprocessivity and a substantially inactive protease the FV PR-RT gene, ornucleic acid, can be mutagenized to create a substitution, deletion orinsertion in the protease sequence. In a specific embodiment, anAspartic acid (Asp, D) residue in the protease catalytic site wasreplaced by an Alanine (Ala; A) residue by altering the codon thatencodes the Aspartic acid residue. This can be accomplished, forexample, by site-directed mutagenesis using the Amersham technique(Amersham mutagenesis kit, Amersham, Inc., Cleveland, Ohio) based on themethods of Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985); Tayloret al., Nucl. Acids Res. 13: 8764-8785 (1985); Nakamaya and Eckstein,Nucl. Acids Res. 14: 9679-9698 (1986); Dente et al., DNA Cloning, Glovered., IRL Press, p. 791-802 (1985); using a Promega kit (Promega, Inc.,Madison, Wis.); or using a BioRad kit (BioRad, Inc., Richmond, Calif.),based on the methods of Kunkel (Proc. Natl. Acad. Sci. USA 82:488-492(1985); Meth. Enzymol. 154:367-382(1987); U.S. Pat. No. 4,873,192), allof which are incorporated herein by reference.

Site directed mutagenesis can also be accomplished using PCR-basedmutagenesis such as that described by Zheng bin et al. (in PCR Methodsand Applications, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., pp. 205-207 (1992), Jones and Howard (BioTechniques 8:178-183(1990); Biotechniques 10:12-66 (1991); Ho et al., Gene 77: 61-68(1989). Other methods of mutagenizing a FV PR-RT gene to substantiallyreduce protease activity, including chemical and radiation methods, areknown to the skilled artisan and considered within the scope of thepresent invention.

The invention also relates to fusion proteins which comprise the FVPR-RT of the invention. Such fusion protein can comprise, for example,the FV PR-RT and (a) a leader amino acid sequence incorporated to directsecretion of the polyprotein out of a host cell, (b) an amino acidsequence added to aid in purification of the FV PR-RT polyprotein, e.g.,a polyhistidine sequence, or (c) additional polypeptides of the foamyvirus Pol open reading frame including the Integrase and/or RNaseH. TheIntegrase and/or RNaseH polypeptides can be active or functionallyinactivated.

The FV PR-RT polyprotein of the present invention can be isolated andpurified by standard methods, including chromatography (e.g. ionexchange, affinity, sizing or high pressure liquid chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of protein. The functional properties canbe evaluated using any suitable assay as described herein or otherwiseknown to the skilled artisan. The polyprotein can also be synthesized bystandard chemical methods known in the art (see, e.g., Hunkapiller etal., Nature 310:105-111 (1984); Stewart and Young, Solid Phase PeptideSynthesis, 2^(nd) ed., Pierce Chemical Company, Rockford, Ill. (1984)).

The isolated FV PR-RT polyprotein having high processivity andsubstantially reduced protease activity produced by any of the abovedescribed methods can be used to prepare cDNA from RNA by, for example,hybridizing an oligo (dT) primer or other complimentary primer with theRNA. The synthesis of a complete cDNA can be accomplished by adding theFV PR-RT and all four deoxynucleotide triphosphates. Using the FV PR-RTof the present invention allows for high processivity. In a specificembodiment, the expressed FV PR-RT was highly active and processednucleic acid sequences longer than that produced by wild-type HIV-1 in asimilar assay.

In addition, the isolated FV PR-RT of the present invention has a highfidelity of transcription of a template sequence. “High” in the contextof the present invention is intended to mean that the isolated FV PR-RTof the present invention has an error rate of at least two-fold lowerthan the HIV-1 reverse transcriptase in the same fidelity assay. In aspecific embodiment of the present invention, the FV PR-RT has a errorrate of about five-fold less than that measured for HIV-1 RT. A standardfidelity assay is described, for example, in Boyer and Hughes, (J.Virol. 74:6494-6500 (2000) incorporated herein by reference). Thefidelity assay involves the copying of a DNA segment that encodes theα-complementing peptide of E. coli β-galactosidase. Single-strandedtemplate DNA is isolated from a bacterial strain that incorporatesdeoxyruracil into DNA, such as plasmid Litmus 29 (Not) from a Dut⁻ E.coli strain. The template U-DNA is hybridized to a primer, which isextended in vitro by the RT to be tested, i.e., FV wild-type or FVmutant RT, or HIV-1 RT, and the like, so that the DNA segment encodingthe α-complementing peptide is copied. The resulting double-stranded DNAis digested, and the fragment containing the LacZα coding region isligated to a plasmid and introduced into a Dut⁺ Ung⁺ E. coli strain,which ensures that the DNA strand synthesized in vitro by the RT beingtested is preferentially retained and copied, giving rise to theplasmids subsequently isolated from individual colonies. The transformedE. coli is grown on plates containing an indicator for β-galactosidaseactivity (X-Gal). Colonies that are either white or light blue arecounted and grown up, and the plasmids recovered. Segments encoding theLacZα peptide are sequenced. In this assay, a percentage of mutationsare silent, therefore the method necessarily underestimates the actualerror rate. The lacZα RNA transcript from Litmus 29 (Not) encodes afusion protein. The LacZα peptide is the C-terminal part of the fusionprotein; the N terminus, which makes no functional contribution to LacZαactivity, is derived from the polylinker. A small part of the N-terminalregion of this fusion protein is encoded with the NotI/BamHI fragmentgenerated in vitro by RT. Because this region does not encode afunctional part of LacZα, only mutations that create termination codonsor frameshift mutations will be detected, potentially skewing theresults. Therefore, only mutations are scored within the 174-bp regionfrom the glycine codon GGA, which is the junction point between lacZαand the polylinker and the first termination codon at the end of lacZα.The result of the fidelity assay for FV RT is compared with the resultfor HIV-1 RT to determine whether the RT has a “high” fidelity.

The FV PR-RT of the present invention is ideally suited forincorporation into a kit for the preparation of cDNA from apolynucleotide sequence, i.e., single-stranded DNA, RNA and the like.Kits are well known in the art and typically can comprise a containermeans being compartmentalized to receive a close confinement therein,such as vials, tubes, and the like, each container means comprising oneof the separate elements of the method used to prepare cDNA from apolynucleotide sequence. For example, there can be provided a containermeans containing FV PR-RT polyprotein in solution. Further, containermeans can contain suitable buffers, substrates for DNA synthesis such asdeoxynucleoside triphosphates, oligo(dT) or complementary primer, andcontrol RNA for use as a standard.

The following examples are illustrative and are not intended to belimiting of the methods or compositions of the present invention. Anysuitable modifications and adaptations which are obvious to one ofordinary skill in the art are within the scope of the present invention.

EXAMPLES

The present example describes the construction of a mutant prototypefoamy virus reverse transcriptase having a methionine in place of avaline in the catalytic site of the enzyme. Further, the exampleprovides the construction of a mutant polymerase-reverse transcriptasemutant fusion protein comprising a mutation in the active site of thepolymerase both with and without the mutation in the catalytic site ofthe reverse transcriptase. These constructs were used to transfect hostcells and demonstrate the overproduction of the gene products. The PR-RTfusion protein comprising an inactive protease and wild-type reversetranscriptase was demonstrated to be not only highly active andprocessive, but also to produce a RT of high fidelity. The mutant PR-RTpolyprotein comprising the protease with substantially reduced activityand mutated reverse transcriptase was not as active as wild-type reversetranscriptase, but was still more active than the RT of many otherretroviruses, including HIV-1 reverse transcriptase.

Materials and Methods

Construction of Prototype Foamy Virus (PFV) RT-V313M Mutant. The shuttlevector pL2-Sub2 (Baldwin and Linial, J. Virol. 73:6387-6393 (1999)) wasused to change the valine residue of the Tyr Xaa Asp Asp (YXDD; SEQ IDNO: 17) motif of SFVcpz(hu) (PFV) reverse transcriptase catalytic siteto a methionine. (Tyr Val Asp Asp (YVDD; SEQ ID NO: 17) to Tyr Met AspAsp (YMDD; SEQ ID NO: 17). The mutagenic oligonucleotide polV313M wasdesigned as a forward primer to change nucleotides 1682-1684 from GTT toATG to create the Valine (V) to Methionine (M) mutation, and changenucleotides 1694-1696 from TAT to TAC to create a unique AflII site tobe used for screening (5′-CTAATGTACAAGTGTATATGGATGATATATACTTAAGCCATG-3′; SEQ ID NO: 1). The reverse primer Int(−)corresponds to nucleotides 2440-2459 of pL2-Sub2(5′-CCCCAGGCTTTACACTTTATG-3′; SEQ ID NO: 2). Using these primers and thepL2-Sub2 template, PCR mutagenesis was used to generate a 794 base pair(bp) fragment that was digested with BsrGI and XbaI and cloned back intothe pL2-Sub2 vector. Clones were screened with AflII to indicate thepresence of the mutated insert and two were identified. Appropriatesegments from these two clones were moved back into the full-length PFVmolecular clone context of pHFV13 by PacI/SwaI digestion and which weresequenced to verify the presence of the mutations. The resultingplasmids were named PFV RT-V313M(A) and RT-V313M(B).

Tissue Culture Methods. FAB cells (BHK cells containing HFV LTR-β-GalDNA)(Yu and Linial, J. Virol. 67:6118-6624 (1993) and 293T cells weregrown in Dulbecco modified Eagle (DME) medium with 10% fetal calf serum.Transfections of FAB cells were performed using LIPOFECTAMINE reagent(GIBCO-BRL, Gaithersburg, Md.) according to the manufacturer'sinstructions (Yu et al., J. Virol. 73:1565-1572 (1999), incorporatedherein by reference). Briefly, FAB cells were plated at a density of9×10⁵ in a 10 cm-diameter tissue culture dish. The next day, cells weretransfected according to the manufacturer's instructions. Briefly, thetransfection mixture contained 25 μl LIPOFECTAMINE reagent and 10 μg ofplasmid DNA. Lipid/DNA complexes were left on the cells 3 hrs, thenremoved and replaced with fresh media. Transfection of 293 cells wasperformed using a Calcium Phosphate method (Chen and Okayama, Mol. Cell.Biol. 7:2745-2752 (1987)).

Cell-free viral supernatants were obtained by filtration of supernatantsthrough a 0.2 μm pore size filter. Intracellular virus was released bycell scraping, 3 freeze/thaw cycles, sonication for 15 sec, andcentrifugation at 2000×g for 10 min. Virus were sedemented through a 20%sucrose cushion containing standard buffer (SB; 100 mM NaCl, 10 mM Tris,1 mM EDTA, (pH 8.0), 20% sucrose) by centrifugation at 24,000 rpm at 4°C. for 2 hr. Pellets were resuspended in SB with 10 mM MgCl₂ andDNase-treated with RQ-1 RNase-free DNase (Promega, Madison, Wis.) (1 μlper 50 μl sample volume) at 37° C. for 1 hr. For polymerase assays,viral supernatants were concentrated to 1/100 (1%) the original volumeusing Amicon CENTRIPREP C-50 spin columns.

A panel of reverse transcriptase inhibitors (Table 1) was tested at aseries of concentrations and cell viability was analyzed for each at 24hours post-treatment by trypan blue staining. The inhibition experimentswere performed with the highest inhibitor concentration that did notaffect cell viability. For pre-treatment with RT inhibitors, FAB cellswere treated with inhibitor for 4 hrs and then infected with cell-freevirus stocks suspended in medium containing inhibitor. Virus was left onthe cells for 24 hrs, and the medium was removed and replaced with freshmedium containing inhibitor. The cells were fixed 48 hrs after infectionand stained for β-gal activity. For post-treatment with RT inhibitors,FAB cells were infected with cell-free virus stocks suspended in mediumwithout inhibitor. Twenty four hrs after infection, the medium waschanged to fresh medium containing inhibitor and 48 hrs post-infection,virus was harvested and assayed for infectivity on fresh FAB cells (Yuand Linial, J. Virol. 67:6618-6624 (1993)).

Nucleic Acid Extractions. Extraction of DNA from viral particles wasperformed as previously described (Yu et al., J. Virol. 73:1565-1572(1999)). Briefly, resuspended pellets were DNase treated, lysed withsodium dodecyl sulfate at a final concentration of 0.5%, extracted twicewith a 24:24:1 mixture of phenol:chloroform:isoamyl alcohol, and nucleicacids were precipitated with ethanol. Pelleted nucleic acids wereresuspended in the original volume of dH₂O and treated with RNase A(Sigma) at 37° C. for 1 hr. The sample was then re-extracted asdescribed and resuspended in the original volume of dH₂O.

Extraction of RNA from viral particles was performed by adding an equalmixture of phenol and 4 M guanidinium isothiocyanate at a 2:1 ratio tothe concentrated virus sample. Samples were extracted twice withchloroform-isoamyl alcohol (24: 1) and nucleic acids were precipitatedwith ethanol in the presence of 10 μg of carrier glycogen (RNase-free;Boeringher Manheim). Pelleted nucleic acids were resuspended in theoriginal volume of diethyl-pyrocarbonate (DEPC)-treated dH₂O and treatedwith RQ-1 RNase-free DNase I (Promega) at 37° C. for 1 hr. The samplewas then re-extracted as described and resuspended in the originalvolume of DEPC-treated dH₂O.

Extraction of genomic DNA was performed by scraping transfected cellsinto PBS, centrifuging the cell suspension at 2000×g for 10 min,resuspending the cells in PBS, and repeating the centrifugation. Cellswere then resuspended in Digestion Buffer made with fresh proteinase Kand RNase A (DB; 100 mM NaCl, 10 mM Tris-Cl (pH 8.0), 25 mM EDTA(pH8.0), 0.5% SDS, 100 μg/ml proteinase K, 1 μg/ml RNase A) andincubated at 50° C. overnight. 1M sodium perchlorate was added at halfthe sample volume, and the sample was phenol-chloroform extracted twice,followed by one chloroform extraction, as described above. The DNA wasethanol precipitated and resuspended in dH₂O.

Reverse Transcriptase-PCR, Cloning, and Sequencing of Revertants. Viralsupernatants were pelleted by ultracentrifugation through a 20% sucrosecushion and viral RNA was extracted as described above. RNA wassubjected to RT-PCR using forward primer pol1441+(5′-CCAACACTCTGCTGGTATTTTAGCTACTA-3′; SEQ ID NO: 3) and reverse primerpol2351-(5′-CAGCTGACAAATTTGGACGTCCG-3′; SEQ ID NO: 4). PCR reactionmixtures contained 2.5 U of avian myeloblastosis virus reversetranscriptase (RT) (U.S. Biochemicals, Cleveland, Ohio), 6 U of RNaseinhibitor (Boerhinger Mannheim, Indianapolis, Ind.), 1×PCR buffer(Perkin-Elmer, Branchburg, N.J.), 1.5 mM MgCl₂, 0.1 mM dNTP mix(Gibco-BRL, Grand Island, N.Y.), 1 U of Platinum Pfx DNA Pol(Gibco-BRL), and 4 ng of each primer. Samples were incubated at 42° C.for 45 min, followed by denaturation for 2 min at 95° C. before thermalcycling was done. Temperatures for denaturing, annealing, and extensionwere 95° C., 50° C., and 72° C. respectively for 45 sec, 45 sec, and 1min each for 35 cycles. The final extension reaction was performed for10 min at 72° C. RT-PCR products were purified using QIAEX II (Qiagen)and A-overhangs were added to the blunt ended products using 5 UPLATINUM TAQ (Gibco-BRL), 1×PCR buffer (Perkin-Elmer), 1.5 mM MgCl₂, and0.1 mM dATP (Gibco-BRL) at 65° C. for 30 min. The DNA products were thencloned into the pGEM T EASY vector (Promega) and blue-white screeningwas used to identify clones containing the insert. White colonies werepicked and sequenced using forward primer pol1548(5′-GGTTAACAGCATTTACCTGGCAAG-3′; SEQ ID NO: 5).

Virion-associated RT assays were performed using viral supernatantscollected 4 days post-transfection of FAB cells and concentrated withCENTRIPREP 50 spin columns (Amicon). The substrate used in these assayswas poly(A):d(T)₁₀ (Sigma). Concentrated viral supernatants were addedto an RT cocktail containing final concentrations of 40 mM Tris-HCl(pH8.0), 50 mM NaCl, 0.5 mM MnCl₂, 15 mM dTT, 25 mM each of dATP, dCTP, anddGTP, 0.1% NP-40, 2 μg/ml poly(A):d(T), and 0.25 μl/ml [α-³²P]-dTTP.Reactions were incubated at 37° C. for 90 min, taking time points at 30min intervals. At each time point, 25 μl of the reaction was spottedonto DE81 filters and allowed to dry. Filters were washed 4 times atroom temperature with 2×SSC for 5 min each, followed by 2 washes with95% EtOH. Filters were then dried and counted in scintillation fluid.

Western Blots. Viral supernatants were concentrated as described abovefor the virion-associated RT assay. Concentrated virus was then added toSDS Sample Buffer and fractionated on a 10% SDS-PAGE gel. Proteins weretransferred to an IMMOBILON-P membrane (Millipore, Bedford, Mass.) andreacted with antibodies according to standard protocols. The membranewas developed using ECL reagents (Amersham Pharmacia), and exposed tofilm.

2-LTR Circle—PCR. 100 ng of DNA was subjected to PCR using primers 350R(5′-AGAAGGGTCCATCTGAGTCAC-3′; SEQ ID NO: 6) and 546F (5′-GATTAAGGTATGAGGTGTGTGG-3′; SEQ ID NO: 7). Reaction conditions were 1×PCR buffer(Perkin-Elmer), 1.5 mM MgCl₂, 0.2 mM dNTP mix (Gibco-BRL), and 1 U TaqPolymerase. Samples were denatured at 95° C. for 5 min, followed by 30cycles of denaturation, annealing, and extension at 95° C., 55° C., and72° C. for 30 sec, 30 sec, and 1 min respectively. A final extension wascarried out at 72° C. for 10 min. PCR products were fractionated on a0.8% agarose gel and the gel was subjected to Southern Blotting asdescribed below.

Southern Blots. 10 μg of DNA was digested with 40 Units of therestriction enzyme NcoI (New England Biolabs) overnight. The digestedDNA samples were fractionated on a 0.9% agarose gel. The gel was washedand transferred to the Hybond membrane following standard protocols. Themembrane was then UV cross-linked and pre-treated with HybridizationBuffer for 2 hrs at 42° C. (HB; 6×SSPE, 0.1% SDS, 20% Formamide, 100ng/ml sheared salmon sperm DNA). Radiolabeled probe to the LTR regionwas generated with the PRIMEIT Random Priming Kit (Stratagene) using theBstEII-pHSRV13 fragment containing the LTR region. Membranes wereincubated with radiolabeled probe in HB at 4° C. overnight. Blots wererinsed and exposed to film.

Construction of Prototype Foamy Virus Reverse Transcriptase ExpressionClones. The construct RT2 pET16b (Kogel et al., Virology 213:97-108(1995)) contains part of the PFV RT coding region but is missing theprotease coding region and most of the RNase H domain. RTVM pET16b issimilar except that the mutation V313M changes the polymerase activesite motif from Tyr Val Asp Asp (YVDD;SEQ ID NO: 17) to Tyr Met Asp Asp(YMDD, SEQ ID NO: 17).

PCR amplification was used to generate DNA fragments containing theprotease coding region and the RNaseH domain that could be linked to thePFV polymerase domain. The PCR amplification of the protease codingregion used a 5′ primer that generated an NcoI site at the ATGinitiation codon (5′-GCGGCGCCATGGCGAATCCTCTTCAGCTGTTACAGCCGCTTCCGGCGG-3′; SEQ ID NO: 8). The introduction of the NcoI siteconverted the start of the protease amino acid sequence from Met Asn ProLeu Gln - - - (MNPLQ - - - ; SEQ ID NO: 18) to Met Ala Asn Pro LeuGln - - - (MANPLQ - - - ; SEQ ID NO: 19). The 3′ primer in the PCRamplification spans a unique AftIII restriction enzyme recognitionsequence in the segment encoding the polymerase domain of PFV RT(5′GCGGCGCCTTGAGGAAGACGTGTCCAA CAATACTGTTTACC-3′; SEQ ID NO: 9). Thisset of PCR primers was used for two separate PCR amplifications usingdifferent substrates: (i) one amplification used pHFV13 (Lochelt et al.,Virology 184:43-54 (1991)), which is a full length, wild-type PFV clone;(ii) the second PCR amplification used PFV protease inactive (Konvalinkaet al., J. Virol. 69:7264-7268 (1995)). The PCR products from these twoamplifications were digested with NcoI; the 3′ end remained blunt fromthe PCR amplification. The resulting 840 bp fragment was cloned as anNcoI/blunt end insert into NcoI/AftIII PFV and NcoI/AftIII Pfv D/A.

The RNaseH domain was also obtained by PCR amplification. The 5′ primerin the amplification spanned a unique PflMI restriction endonucleaserecognition site in the PFV pol domain (5′ GCGGCGGGATCCGCTTTACCCATTAGTGGATAACATGGAT GAC 3′; SEQ ID NO: 10). The primer also generated a BamHIsite 5′ of the PflMI site. The 3′ primer added a TAG termination codonafter the tyrosine codon that normally marks the last amino acid in theRNaseH domain and also generated an EcoRI site 3′ of the terminationcodon (5′ GCGGCGGAATTCGCGCTAATATTGTTTGGGATATCCTTTTATATAAT GACCCTG 3′;SEQ ID NO: 11). The underlined sequence is a unique EcoRV restrictionendonuclease recognition sequence present in the coding region. The PCRfragment was digested with BamHI/EcoRI and cloned into BamHI/EcoRIdigested LITMUS-29. The clone, designated 3′ PFV, contained the normalC-terminus of the PFV RNaseH domain.

To simplify protein purification, the C-terminus was further modified bythe addition of six histidine residues before the termination codon. Theunique EcoRV site was used as an entry point. The clone 3′ PFV wasdigested with EcoRV and EcoRI, then ligated to synthetic DNA fragmentsto construct the clone 3′ PFV (His). The synthetic DNA fragments weregenerated by kinasing oligonucleotide 1 (5′-ATCCCAAACAATATTCTTCCCATCATCACCACCATCATTAGTAGGTACCCG-3′; SEQ ID NO: 12) and oligonucleotide2 (5′-AATTCGGGTACCTTACTAATGATGGTGGTGATGATGGG AAGAATATTGTTTGGGAT-3′; SEQID NO: 13), followed by heating and slow cooling the oligonucleotides toallow them to anneal. The C-terminus of PFV RT is normally - - -ProLysGlnTyr ( - - - PKQY; SEQ ID NO: 14). In PFV (His) the codingregion was altered so that the C-terminus wasProLysGlnTyrProSerSerGlyHisHisHisHisHisHis ( - - - PKQYPSSGHHHHHH; SEQID NO: 15).

Three oligonucleotide fragments were ligated to generate clonescontaining all of the protease as well as the entire polymerase andRNaseH domains. The fragments were: a) the 840 bp NcoI/AftIII fragmentfrom either NcoI/AftIII Pfv or NcoI/AftIII PFV D/A, which contain theactive or inactive protease coding region respectively, b) theAftIII/PflMI fragment from either RT2 pET16b or RTVM pET16b, whichcontain the wild-type or V313M active site in the polymerase domain, andthe PflMI/EcoRI fragment from 3′ PFV (His). The fragments wereco-ligated into NcoI/EcoRI digested LITMUS-29 to generate four clones:a) PFV (His), which has a wild-type protease and a wild-type RT; b) PFVD/A (His), which has an inactive protease and a wild-type RT; c) FVRTV313M (His), which has an active protease and the V313M mutation inthe RT; and d) FV D/A RTV313M (His), which has an inactive protease andthe V313M mutation in RT. For protein expression, the inserts werecloned into NcoI/EcoRI digested pT5m, which is similar in concept to thepET vectors. The clones were transformed into the Rosetta E. coli strain(Novagen, Madison, Wis.), a BL21 derivative. Only the clones that hadthe inactive protease expressed significant levels of protein in thissystem.

Protein Expression and Purification. Bacteria were grown at 37° C. withagitation to an O.D.600 nm of 0.5 to 0.6. Expression of the RT proteinwas induced by the addition of 0.2 μM IPTG and incubation of thebacteria for an additional 3 hr before harvesting. Fifty grams ofpelleted bacteria was extracted in 100 ml of 50 mM NaPO₄, pH 8.0, 50 mMNaCl, 1.5 mM PMSF and 0.75 mg/ml lysozyme. The sample was incubated onice for 30 min. 10.75 ml of 4 M NaCl was added to the suspension,followed by 3×30 sec sonication at 90% power (max. Watt 350) and 70%pulse. A ¾-inch probe was used with 5 min between each sonication. Thesuspension was centrifuged at 85,000×g for 90 min and the clear portionof the supernatant was removed. The remaining, somewhat viscous, portionof the supernatant was recentrifuged and the clear supernatant wascollected. Supernatants were diluted 1:1 with 66 mM NaPO₄, pH 6.8, and300 mM NaCl.

A 15 ml Q-Sepharose column and a 15 ml nickel column (Qiagen) werepoured and connected in series with the Q column first. The columns wereequilibrated with 50 mM NaPO₄, pH 7.0 and 300 mM NaCl. Dilutedsupernatants were loaded onto the columns at 1 ml/min. After loading,the columns were washed with equilibration buffer. After 100 ml of flowthrough, the Q column was removed and the nickel column was washed withan additional 150 ml of buffer. The Q column was next washed with 250 mlof 50 mM NaPO₄, pH 6.0, 10 mM imidazole, 300 mM NaCl, and 10% glyerol(w/v). A 150 ml×150 ml, 10 mM to 500 mM imidazole (in pH 6.0 buffer)gradient was used to elute the protein. Eight (8) ml fractions werecollected. Fractions were pooled based on SDS-PAGE analysis. Theresulting pool was between about 60 to 70 ml. This pool was divided inhalf, with each pool dialyzed versus 3×500 ml of 25 mM Tris acid/25 mMTris base. Sample (50% of the pool) was removed from dialysis andcentrifuged at 12,000×g for 30 min. The pellet was resuspended with 3 mlof 20 mM Hepes, pH 7.0, 100 mM imidazole, 300 mM NaCl, 1 mM EDTA and 10mM DTT. One (1) ml of 2 M NaCl was added to the suspension, the samplewas stirred for 30 min, and centrifuged at 12,000×g for 30 min.

The supernatant was loaded onto a 1.6 cm×85 cm Sephacryl 200 column(Amersham-Pharmacia) equilibrated with 20 mM Hepes, pH 7.0, 100 mMimidazole, 300 mM NaCl, 1 mM EDTA, and 1 mM DTT. The column was run at0.2 ml/min with 10 min fractions collected. After gel analysis, thefractions were pooled. The gel filtration runs were then combined,resulting in approximately 20 ml. The sample was then dialyzed versus 25mM Tris acid/25 mM Tris base, 10% glycerol, 1 mM EDTA, 10 mM imidazoleand 1 mM DTT. Sample was removed from dialysis, centrifuged at 12,000×gfor 30 min and loaded on to a 5 ml Q column equilibrated with thedialysis buffer. The column was run with a flow rate of 1 ml/min and 4ml fractions of the flow through was collected and analyzed by SDS-PAGE.Samples were pooled, one-tenth volume of NaCl was added to the pool, andthe samples were concentrated by centrifical ultrafiltration using a 10kD cutoff membrane (Filtron). Sample analysis by SDS-PAGE, CoomassieStain, showed purity greater than 97%. Gel filtration indicated thepresence of a small amount of dimmers, which increased upon storage inthe cold. Yields were generally between about 12 mg and 20 mg. All stepswere done at 4° C.

In vitro Polymerase Assays. The substrate for in vitro polymerase assayswas the M13 −47 sequencing primer annealed to single-stranded M13mp18DNA (New England Biolabs). The −47 primer as end-labeled using T4polynucleotide kinase and [γ³²P]ATP then annealed to the single-strandedM13mp18 DNA. The substrate was resuspended in 90 μl of buffer lackingdNTPs to a final concentration of 2 nM (see below) and 1.0 μg of enzyme.The mixture was incubated at room temperature for 5 min to allow theenzyme to bind the template-primer. The reaction was initiated by theaddition of 10 μl of 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, and 0.2 mMdTTP. In a processivity assay, 0.5 units poly(rC):oligo(dG) was alsoincluded as a cold trap. The final concentrations of buffer in thereactions were: 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl₂, 2.0 mMDTT, 10.0 mM CHAPS, 100 μg/ml acetylated bovine serum albumin (BSA),10.0 μM of each DNTP in a final volume of 100 μl. After 10 min at 37 °C., the reaction was terminated by extraction with an equal volumephenol/chloroform and the mixture was precipitated with isopropylalcohol. The sample was resuspended in 10 μl loading dye and 4 μl loadedon a 6% sequencing gel. The products of the reaction were visualized byexposure to X-ray film. The DNTP curves were performed using the bufferdescribed above and the indicated concentration of dNTPs, in the absenceof poly(rC):oligo(dG). Reactions were incubated at 37° C. for 15minutes.

The 3TCTP inhibition assays were performed using the M13 template andthe −47 primer. The −47 sequencing primer was annealed to the singlestranded M13mp18 DNA by heating to 95° C. and slowly cooling to roomtemperature. The template-primer was extended by adding 1.0 μg of RT in25 mM Tris(pH 8.0), 75 mM KCl, 8.0 mM MgCl₂, 100 μg/ml BSA, 10 mM CHAPS,10 μM each of dATP, dGTP, and dTTP, 2.0 μM [α-³²P]dCTP, and theindicated concentrations of 3TCTP (Moravek Biochemicals, Brea, Calif.)in a 100 μl reaction volume. The mixture was incubated at 37° C. for 30min, then halted by the addition of 3 ml of ice-cold trichloroaceticacid (TCA) and the precipitated DNA was collected by suction filtrationthrough Whatman GF/C glass filters. The amount of incorporatedradioactivity was determined by liquid scintillation counting.

Fidelity Assay. The fidelity assay used was that disclosed by Boyer andHughes (J. Virol. 74:6494-6500 (2000), incorporated herein byreference). Briefly, plasmid Litmus 29 was obtained from New EnglandBioLabs. The plasmid contains an M13 origin of replication and arestriction enzyme recognition site polylinker, including a recognitionsite for BamHI, 5′ of the coding region for the LacZα-complementingfragment. Litmus 29 was linearized with HpaI, ligated to NotI linkers,and recircularized to make Litmus 29 (Not). The new NotI recognitionsequence was located 3′ of the lacZα-coding region.

Single-stranded uracil-containing DNA was formed by introducing theconstruct Litmus 29 (Not) into the Dut⁻ Ung⁻ male E. coli strain CJ236(New England BioLabs). This bacterial strain introduced deoxyuracilresidues into the plasmid DNA during replication. To generatesingle-stranded U-DNA Litmus 29 (Not), the helper phage M13K07 (NewEngland BioLabs) was used according to the manufacturer's instructions.Briefly, 50 ml of Luria-Bertani medium supplemented with uridine (0.25μg/ml) was inoculated with a colony of Litmus 29 (Not) in CJ236. Theculture was incubated at 37° C. with agitation until the solution wasslightly turbid. The helper phage M13K07 was added to a finalconcentration of 10⁸ PFU/ml. The culture was incubated at 37° C. withagitation for an additional 60 min. Kanamycin was added to a finalconcentration of 70 μg/ml, and the culture was incubated overnight at37° C.

Bacteria were removed by sedimentation twice at 8,000 rpm for 10 min.One-fifth volume of 2.5 M NaCl-20% PEG (polyethylene glycol) 6000-8000(NaCl-PEG) was added to the supernatant, and the solution was incubatedon ice for 2 h. The phage particles were isolated by centrifugation at8,000 rpm for 10 min. The pellet was resuspended in 1.6 ml of 10 mMTris-Cl (pH8.0)-1.0 mM EDTA (TE) and divided into two tubes. Thesolution was cleared by centrifugation in a microcentrifuge at fullspeed to remove any trance of bacteria. MgCl₂ was added to a finalconcentration of 10 mM, and DNase was added to the solution to removeboth contaminating bacterial and double-stranded phage DNA released bybacterial lysis. Intact phage particles were isolated by the addition of200 μl of NaCl-PEG solution to each tube and centrifugation in amicrocentrifuge for 5 min at full speed. The phage pellet wasresuspended in 100 μl of TE and extracted three times withphenol-chloroform. After the addition of NaCl to a final concentrationof 50 mM, the phage DNA was precipitated with 1 volume of isopropanol,then resuspended in 400 μl of H₂O and stored at −20° C. The phage DNAcan be further purified by use of a Qiagen M13 purification Kit or otherstandard protocol for the isolation of U-DNA.

The fidelity assay used a fidelity primer (5′-CCCATGGTGAAGCTTGGATCCACGATATCCTGCAGG-3′; SEQ ID NO: 16) which matches the sequencesurrounding the BamHI recognition site in the Litmus 29 (Not)polylinker. For each fidelity assay, 2.5 μl from a 10.0-A₂₆₀/ml stock offidelity primer was annealed to 1.0 μg of single-stranded U-DNA byheating and slow cooling. Each sample was adjusted to consist of 25 mMTris-Cl (pH 8.0), 75 mM KCl, 8.0 mM MgCl₂, 2 mM dithiotreitol, 100 μg ofbovine serum albumin per ml, 10 mM CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 20 μMeach DATP, dCTP, dGTP, and dTTP. One microgram of reverse transcriptaseto be tested (HIV-1 RT, PFV-RT, PFV D/A-RT and PFV D/A-RTV313M) wasadded, and the samples were incubated for 15, 20, 30 min, or longer at37° C. The reactions were stopped by the addition of 1 volume ofphenol-chloroform, followed by isopropanol precipitation with a 70%ethanol wash. The extended template primers were digested with BamHI andNotI, and the resulting fragments were fractionated on a 2% agarose gel.If an RT copied the lacZα portion of the template past the NotIrecognition sequence, a band approximately 300 bp in size was visible inthe gel. Primers that were not extended past the NotI site were annealedto phage DNA that was linearized with BamHI which migrated near the topof the gel. The BamHI/NotI fragment encoding LacZα was isolated from thegel and purified. These fragments were ligated into the B/N RT(His)construct (Boyer and Huges, supra), transformed into E. coli DH5α andplated onto NZY (10.0 g NZ amine, 5.0 g NaCl, 5.0 g yeast extract, and2.0 g MgSO₄ per liter)-ampicillin plates supplemented with5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal). The dark blue,light blue, and white colonies were counted. DNA was isolated from thelight blue and white colonies and tested by digestion with BamHI andNotI. The 300 bp lacZα inserts were than sequenced.

Results

Efficacy of Reverse Transcriptase Inhibitors. The unique timing ofPrototype Foamy Virus (PFV; previously designated SFVcpz(hu)) reversetranscription allows the virus to infect cells that were pre-treatedwith reverse transcriptase inhibitors, since infectious PFV particlesalready contain the viral DNA. However, PFV is unable to produceinfectious particles from cells that are treated with inhibitor. Thisspecific inhibition was demonstrated in previous studies with theinhibitor AZT, however not with the inhibitors 3TC and ddI (Yu et al. JVirol. 73:1565-1572 (1999)). To better understand the susceptibility ofPFV, a more complete panel of HIV-1 RT inhibitors was tested.

In studies designed to measure the effect of adding RT inhibitors totarget cells, FAB cells, which are BHK-derived cells that containβ-galactosidase driven by the FV LTR (Yu and Linial, J. Virol.67:6618-6624 (1993)), were treated with inhibitor 4 hrs prior to theaddition of PFV viral stock. Forty-eight (48) hours after infection,cells were stained with X-Gal staining solution and infectivity of thevirus, as measured by viral titer was determined. The reversetranscriptase inhibitors 3TC, 3′-Azido-2′,3′-dideoxy-5-methylcytidine(AzddMeC), 3′-azido-2′,3′-dideoxyuridine (AzddU), AZT,2′,3′-didehydro-2′,3′-dideoxythymidine (D4T), 2′,3′-dideoxycytidine(ddC), beta-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), andphosphonoformate (PFA) were used in these experiments at the highestconcentrations that demonstrated no cellular toxicity. Allconcentrations used were known to inhibit HIV-1 replication. None of theinhibitors had a dramatic effect on PFV infectivity, as predicted by theFV life cycle (Table 1).

To test the effects of the inhibitors on virus producing cells, FABcells were infected with PFV at a multiplicity of infection (MOI) of 1,and 24 hrs after infection inhibitor was added. Forty-eight (48) hrsafter infection, virus was harvested from these cells, and titered onfresh FAB cells. The titers revealed a range of inhibition, but only AZTdemonstrated a specific decrease in infectivity of greater than 10 fold(Table 1).

It was not surprising that 3TC was unable to inhibit PFV replicationgiven that the highly conserved YXDD (SEQ ID NO: 17) sequence in PFV RTis YVDD which is the same as the sequence of the resistant HIV-1 RTcatalytic site. To determine whether mutating the RT sequence to YMDDmight render PFV sensitive to 3TC, valine (V) was changed to amethionine (M) in the PFV RT YXDD motif to create the mutant PFVRT-V313M.

TABLE 1 Efficacy of Reverse Transcriptase Inhibitors Titer Relative toNo-Drug Control Inhibitor Pretreated Post-treated No drug 100 100 3TC 60μM 120 91 AzddMeC 25 μM 70 13 AzddU 25 μM 51 11 AZT 100 μM 22 <0.14 D4T25 μM 54 32 ddC 25 μM 42 32 FTC 60 μM 122 138 PFA 25 μM 100 23

TABLE 2 Replication of HFV RT-V319M mutant in FAB cells Days post- VirusTiter (flu/ml) transfection WT HFV V319M-CloneA V319M-Clone B Mock 2 1.4× 10³   1 × 10¹   1 × 10¹ <10¹ 4 1.7 × 10⁴   1 × 10¹   5 × 10¹ <10¹ 61.4 × 10⁴   4 × 10¹   8 × 10¹ <10¹ 8 8.7 × 10³   1 × 10¹   3 × 10¹ <10¹10 3.6 × 10² 4.9 × 10² <10¹ 12 5.6 × 10³ 8.8 × 10³ <10¹ 14 1.5 × 10⁵ 1.7× 10⁵ <10¹ 16 1.5 × 10⁵ 1.2 × 10⁵ <10¹ 18 2.9 × 10⁴ 2.0 × 10⁴ <10¹

Replication of RT-V313M. The effect on virus replication of the V to Mmutation in PFV RT was determined by transfecting FAB cells with DNA ofPFV RT-V313M(A) and (B), two independently derived clones. Two daysafter transfection, supernatants were collected and titered on fresh FABcells. Surprisingly, the mutant virus showed an extremely low titer,indicating that very little infectious virus was being made with themutant RT (Table 2). In an attempt to select for second-site mutationsor reversions, the cells transfected with the mutant RTs were passageduntil supernatants showed titers similar to wild-type levels. At two-dayintervals, the supernatants were collected and titered on fresh FABcells while the cells were split 1:3 and maintained. By day 12 the cellsproduced virus with titers similar to wild type PFV (Table 2).

Virus was isolated from the cell-free supernatants of day 14, whentiters of the PFV RT-V313M viruses were at the highest level. RNA wasextracted from virus and used in RT-PCR reactions designed to amplifythe region of PFV pol encoding the V313M mutation. The RT-PCR productswere used to isolate individual clones, which were then sequenced andanalyzed for any changes from the original sequence. The sequencealignment of the individual clones and the original sequence revealed atwo-nucleotide change at the ATG methionine codon to regenerate thevaline codon present in wild type. The clones also had nonspecificsingle nucleotide changes, however none of these individual mutationswere found in any other clones, suggesting that they were not secondsite mutations involved in reversion. Thus, it appears that PFV does notreplicate if the RT has a methionine in the second residue of the YXDDmotif (SEQ ID NO: 17).

Virion-Associated Activity of PFV RT-V313M. The fact that the V313Mmutation is located near the polymerase catalytic site suggested thatthis mutation might affect the catalytic activity of the enzyme. To testwhether PFV RT-V313M had any exogenous polymerase activity, supernatantswere collected from transfected FAB cells. Concentrated virions wereused in an RT assay which measured the incorporation of radiolabelednucleotide onto a polyA:oligo dT primer-template over the course of 90minutes. As a positive control for this assay, another mutant virus,PFV-IN(−), which contains a wild type RT enzyme but has a mutation inthe active site of Integrase (IN) making it replicative defective, wasused. PFV-IN(−) is unable to produce infectious virus, similar to PFVRT-V313M, and therefore produced similar amounts of virus forcomparison. A Western blot was performed on the IN(−) and the V313Mconcentrated virus particles to demonstrate that enzyme from similarnumbers of viral particles were used in the assays. The plasmid pNEB193,which contains no viral sequences, was used as a negative control fornormalization of the assays. The results from the RT assays demonstratethat the V313M mutant RT retains approximately 50% of wild typepolymerase activity (FIG. 2). Given the dramatic effect this mutationhad on replication, it was surprising to find a relatively modestdecrease in RT-V3 13M activity.

RT-V313M Reverse Transcription Products in Cells. Since the mutant RTretained substantial polymerase activity, it was determined whether ornot reverse transcription was completed in cells transfected with PFVRT-V313M. Because the PFV RT-V313M mutant did not replicate well, it wasdifficult to obtain significant quantities of virus or viral products incells that were transfected using LIPOFECTAMINE. To increase theproduction of mutant viruses in transient transfections, the viralgenome was placed under the control of the CMV promoter. Calciumphosphate transfection was used to introduce the CMV-driven viral clonesinto 293T cells. This technique greatly increased viral expression andsimplified the analysis or replication-defective viruses.

Two different approaches were used, first was the detection of 2LTRcircles using PCR combined with Southern blot analysis. 2LTR circles area by-product of the completion of reverse transcription of thefull-length linear viral DNA. A fraction of the full-length DNAs arejoined by blunt-end ligation to form 2LTR circles. Primers were designedto hybridize to the 5′ and 3′ LTR regions such that polymerization wouldextend the primers to the ends of unintegrated linear viral DNA. If thecDNA is in a linear form or incomplete, no productive PCR product wouldbe generated. However, when 2LTR circles are present, a specific PCRproduct is formed. Two positive controls containing wild type RTs wereused including the IN(−) mutant described above and the FST4 mutant,which lacks the Env cleavage site. Neither mutant produces infectiousvirus, but both release particles that contain wild-type RT. The PolΔ5mutant, which contains a large 500 bp deletion in the RT coding region,does not have a functional RT enzyme and consequently does not replicatein the RT coding region, was used as a negative control.

293T cells were transfected with the above described mutants and fourdays post-transfection genomic DNA was isolated. PCR products from 500ng of cellular DNA were separated on agarose gels and transferred to anylon membrane for Southern hybridization. A radiolabeled fragment ofthe PFV LTR region was used as the probe. The Southern blot showed thatthe PCR product specific for the 2LTR circle was present in all of thesamples derived from transfections of viruses with a wild-type RT enzyme(WT, IN(−), and FST4), but was absent in samples derived from virus withthe RT deletion (PolΔ5) and the RT-V313M virus. Since the positivecontrol virus FST4 had a weaker signal than the wild type and IN(−)controls, the FST4 virus was used to determine the range of sensitivityfor this assay. The genomic DNA from cells transfected with the FST4virus was serially diluted 1000-fold (500 ng to 0.5 ng) and thosesamples were subjected to the PCR and Southern blot described above.2LTR circles could be detected in a 100-fold dilution of the originalsample, indicating that the production of 2LTR circles is at least100-fold lower with the V313M mutant RT than with the wild type RT.

The second approach employed to address whether or not the RT-V313Mmutant was able to complete reverse transcription involved directSouthern blotting of the genomic DNA isolated from transfected 293Tcells. The genomic DNA samples used in the 2LTR PCR experiments weredigested with the restriction enzyme NcoI. This enzyme produces DNAfragments that allowed the distinction between the input plasmid DNA andthe desired linear viral cDNA. A diagram of NcoI sites in the viralplasmid used for transfection and predicted sizes of resulting productsthat hybridize to the probe is provided as FIG. 3. After digestion, theDNA samples were fractionated on an agarose gel, transferred to a nylonmembrane, and probed with the same radiolabeled PFV-LTR fragment usedabove. Detection of a 2.2 kb band corresponding to the 3′ LTR of thecDNA product indicated the presence of viral cDNA in the transfectedcells (FIG. 3). The blot demonstrated that cells transfected with eitherof the viruses that contain wild-type RT enzyme (WT and IN(−)) wereundergoing reverse transcription and generated detectable amounts ofcDNA. However, cDNA could not be detected in cells transfected with thePolΔ5 or V313M viruses. These results agree with the 2LTR results anddemonstrate that although the RT-V313M has about 50% activity in anexogenous RT assay, it was unable to complete reverse transcription inan infected cell.

RT-V313M Activity in Vitro. In order to perform more detailedcharacterization of RT-V313M RT activity, a His-tagged version of theprotein was produced in E. coli and purified over a nickel column asdescribed above. In the context of the virus, reverse transcriptase isexpressed as part of a larger Pol polyprotein, which also contains IN(integrase) and PR (protease). Unlike most other retroviruses, the foamyvirus Pol undergoes a single cleavage event, which releases IN, leavinga PR-RT fusion protein. However, in some bacterial over-expressionsystems a second cleavage event between PR and RT has been reported(Pfrepper et al., J. Virol. 72-7648-7656 (1998)). To avoid this cleavageand the toxicity of PR in bacteria, the PR-RT fusion protein expressedin bacteria had a point mutation in the PR active site that inactivatedthe protease (Konvalinka et al., J. Virol. 69:7264-7268 (1995)).Expression plasmids with the mutation in the protease active site aredesignated D/A.

Using the purified recombinant PFV enzymes along with purifiedrecombinant HIV-1 RT, it was first determined whether the FV RT-V313Mwas sensitive to 3TC. The M13 template and primer were annealed and thenincubated with either FV D/A-RT, FV D/A-RT V313M, or HIV-1 RT andincreasing concentrations of 3TCTP. Samples were TCA precipitated, boundto glass filters, and incorporation of radiolabeled dCTP was measured.In the absence of 3TCTP, it was found that both the V313M RT and theHIV-1 RT displayed about 35% polymerase activity of FV D/A RT. This wassimilar to the decrease in virion-associated RT activity observed forthe V313M mutant virus described above. These levels of RT activity inthe absence of drug were set at 100% activity for each recombinant RT.HIV-1 RT demonstrated sensitivity to 3TC, retaining only 37% of itsactivity at 1.0 μM 3TCTP (FIG. 4).

To compare the RT activities of the mutant and wild-type FV enzymes,standard kinetic analyses were performed using homopolymeric templates.However, the results obtained from these experiments suggested that ahomopolymeric template may not be the appropriate substrate to studythese enzymes. As an alternative method to compare the mutant andwild-type FV RTs, the effects of dNTP concentration on polymerizationusing a heteropolymeric template were examined. The M13 single strandedDNA template and radiolabeled primer were annealed, incubated witheither HIV-1 RT, FV D/A-RT, or FV D/A-RTVM, and a reaction mixturecontaining increasing concentrations of each dNTP. The reaction wascarried out for 15 min and then fractionated on a 6% sequencing gel. Atconcentrations above 1.0 μM most of the DNA synthesized by FV D/A-RT wastoo large to be measured accurately on a sequencing gel. The mutant FVD/A-RTVM was severely impaired in its polymerization at the lowest dNTPconcentrations. However, the mutant did synthesize DNAs of 100 nt orlonger at dNTP concentrations of 0.2 μM or greater and does not approachsaturation until the highest dNTP concentrations. These resultssuggested that the k_(m) of the V313M mutant might be in the about 5 toabout 10 μM range. HIV-1 RT also approached saturation at the highestdNTP concentrations, which suggested a k_(m) also in the about 5 toabout 10 μM range, similar to the V313M RT mutant. To measure the sizeof the DNA products synthesized by FV D/A-RT more accurately, anotherpolymerization assay was performed using an M13 template and theproducts were fractionated on a 1% alkaline agarose gel. DNAs up to 7 kbwere synthesized in 20 min, but the rate of DNA synthesis was stillincreasing at dNTP concentrations greater than 20 μM. These datasuggested that the k_(m) of FV D/A-RT for dNTPs was higher than that ofthe mutant.

Because FV RT-V313M did not support the synthesis of full-length cDNAand the recombinant FV D/A-RTV313M protein demonstrated a significantdecrease in primer extension, particularly at low dNTP concentrations,the processivity of the mutant FV enzyme was measured and compared toboth wild type FV D/A-RT and HIV-1 RT. The −47 primer was end-labeledand then annealed to the single-stranded M13mp18 DNA. This radiolabeledprimer-template was incubated with the purified HIV-1 RT, FV D/A-RT andFV D/A-RTV313M proteins to allow the enzyme to bind the primer-template.The reaction was initiated by the addition of all of the four dNTPs. Insome reactions an unlabeled poly(rC):oligo(dG) cold trap was added. Thecold trap was added in excess, and bound any RT proteins thatdissociated from their original primer-template. Thus, the RT extensionproducts produced in the presence of a trap indicated how far the enzymewas able to extend before it dissociated from the template.

The reactions were carried out for 10 minutes, followed by immediatephenol:chloroform extraction and isopropanol precipitation of thenucleic acid. The samples were then fractionated on a 6% sequencing gel.In the absence of the cold trap, the HIV-1 RT generated products ofabout 350 to about 600 nucleotides in length while the FV D/A RTgenerated products well above 600 nucleotides in length. The mutant FVD/A-RTV313M generated products of an intermediate length. In thepresence of the cold trap, the HIV-1 RT demonstrated predominantlyshorter products, however, the FV D/A-RT still generated mostly productslonger than 600 bp. In contrast to the wild type FV RT the FVD/A-RTV313M enzyme did not generate products longer than 600 nt, butinstead showed a range of shorter products, none of which were longerthan 400 bp. These results demonstrated that wild type FV RT was ahighly processive enzyme, producing large quantities of long productsafter only 10 minutes in the assay used. The inability of the FVD/A-V313M RT to generate longer products indicated that this enzyme wasdramatically less processive than the wild type FV enzyme, although itwas similar to HIV-1 RT. The substitution of methionine for valine inthe YXDD motif affected processivity which might explain the inabilityof the mutant RT to complete reverse transcription. Foamy virus Reversetranscriptase appears to require valine in its catalytic site in orderto maintain processivity, and consequently its ability to successfullycomplete reverse transcription. In addition, it appears that the FV RThas about a five-fold lower error rate than the rate that has beenmeasured for HIV-1 RT.

DISCUSSION

The highly conserved YXDD (SEQ ID NO.: 17) motif contains two of thethree aspartic acid residues that make up the RT active site (Julias, etal., J. Virol 75:6537-6546 (2001); Kohlstaedt et al., Science256:1783-1790 (1995); Nanni et al., Perspect Drug Discov. Des. 1:129-150(1993); Tantillo et al., J. Mol. Biol. 243:369-387 (1994)). Thethree-dimensional crystal structure of HIV-1 RT shows that the YXDDmotif is located on the β9-β10 hairpin and is part of the dNTP bidingsite (Huang et al., Science 282:1669-1675 (1998); Jacobo-Molina et al.,Proc. Natl. Acad. Sci. USA 90:6320-6324 (1993)). More specifically, thesecond variable residue (X) appears to interact with the ribose of theterminal primer nucleotide (Gao et al., J. Mol. Biol. 300:403-418(2000); Sarafianos et a., Proc. Natl. Acad. Sci. USA 96:10027-10032(1999); Tantillo et al., J. Mol. Biol. 243:369-387 (1994)). It has beenpostulated that this interaction between the second (X) residue and theprimer may be important for affinity to the template-primer, anddifferent residues in the second position may alter flexibility of thedNTP binding pocket, leading to changes in the behavior of the enzyme(Boyer and Hughes, Antimicrobial Agents and Chemotherapy 39:1624-1628(1995); Ding et al., Biopolymers 44:125-138 (2000); Gao et al., J. Mol.Biol. 300:403-418 (2000); Harris et al., J. Biol. Chem. 273:3324-3334(1998); Sarafianos et al., Proc. Natl. Acad. Sci. USA 96:10027-10032(1999); Tantillo et al., J. Mol. Biol. 243:369-387 (1994)). Therefore,it is likely that the size and/or the hydrophobicity of the amino acidside chain of the second residue in the YXDD motif can influence thecatalytic activity of the polymerase RT. Changes in the YXDD motif canalso alter the active site structure and/or lead to the repositioning ofthe template-primer in such a way that the RT/DNA complex incorporatesnormal dNTPs less efficiently. Such an altered structure could be lessstable, and thus PFV RT-V313M could dissociate more easily from itsnucleic acid substrate than the wild-type enzyme, reducing processivity.

In contrast to PFV, HIV-1 with the 3TC resistance mutation YVDD (M184V)replicates almost as well as wild-type (YMDD) (Schinazi et al.,Antimicrobial. Agents and Chemotherapy 37:875-881 (1993)). In vitro,HIV-1 M184V RT displays 75% of the activity of wild type RT and hasreduced procesivity (Boyer and Hughes, Antimicrobial. Agents andChemotherapy 39:1624-1628 (1995)). Further, MLV with the mutant YMDDmotif (V223M) retains 20% of wild type activity yet it is still able toreplicate with only a 10-fold decrease in titer compared to wild type(Halvas et al., J. Virol. 74:312-319 (2000)). In vitro, recombinant wildtype and YMDD MLV RTs show similar polymerase activities and havesimilar extension abilities (Boyer et al., J. Virol. 75:6321-6328(2001)). Thus, although mutations in the YXDD motif affect thepolymerase activities of the HIV-1 and MLV, these viruses are still ableto replicate with either M or V present in the variable position of theYXDD motif; the architecture of the active site for both HIV-1 and MLVmust be able to tolerate either amino acid residue. The data providedabove demonstrates that the same is not true for PFV RT. A PFV RT withYMDD (V313M) could not support productive replication of the virus. Themutant virus rapidly reverted to YVDD despite the fact that the mutantRT retained approximately 50% of wild type activity.

Conventional retroviruses generate both Gag and Gag-Pol fusion proteins,at a ratio of about 20:1. The result is a virus particle that containsapproximately 50-75 Pol proteins per particle (Vogt and Simon, J. Virol.73:7050-7055 (1999)). PFV Pol is expressed from its own spliced message,and the mechanism for packaging the Pol protein is unclear. In FVinfected cells, the Pol protein is sufficiently abundant that it can beeasily detected by Western blot. However, we have been unable to detectPol in virus particles by Western blot or by radioimmunoprecipitation,although in more recent work using a monoclonal antibody to Pol, it hasbeen detected. There is some evidence for a cis-acting RNA sequence atthe 5′ end of the FV genome (CASI) that is not involved in RNA packagingbut is required for PR activity (Heinkelein et al., J. Virol.74:3141-3148 (2000)) and for Pol packaging. If Pol must bind to aspecific region of the RNA for encapsidation, this would limit thenumber of molecules that can be incorporated.

A low number of Pol molecules in a viral particle places a specialburden on PFV RT, relative to other retroviral RTs. One or two FV RTwould have to accomplish the same task carried out by the 50 to 75 RTspresent in other retroviral particles (Vogt and Simon, J. Virol.73:7050-7055 (1999)). In order to overcome this disadvantage, FV RTwould necessarily be a particularly efficient polymerase, and ourresults suggest that this is the case. It is likely that a moderatedecrease in RT activity in viruses such as HIV-1 and MLV does not have adramatic effect on replication because there are a relatively largenumber of RTs in the particle that can collaborate to carry out reversetranscription (Julais et al., J. Virol. 75:6537-6546 (1999); Telesnitskyand Goff, EMBO J. 12:4433-4438 (1993)). In the case of PFV, a minordecrease in RT activity could be much more detrimental if only one ortwo RTs were responsible for reverse transcription.

As might be expected if PFV requires a highly active RT, the aboveresults also show that FV RT is an extremely processive. In theprocessivity assay, wild type PFV RT was able to generate products thatwere significantly longer than 600 nt in 10 min. HIV-1 RT is much lessprocessive, generating products ranging from 100 to about 400 nt inlength. This high level of processivity for PFV should effectivelycompensate for the small number of Pol proteins in a virion. The aboveresults support the idea, showing that the mutant V313M, which cannotreplicate, has a decrease in processivity compared to wild type PFV RT.Yet, in vitro the mutant enzyme has a processivity that is comparableto, or slightly better than that of HIV-1 RT. Thus, FV is not able toreplicate with a mutant RT that has a processivity similar to that ofHIV-1.

The discovery of the high processivity of isolated PFV reversetranscriptase provides a particularly useful reagent for use inmolecular biology. The enzyme is particularly useful for the in vitroproduction of RNA from a polynucleotide sequence. For example the enzymecan be used in place of typical reverse transcriptase in PCR kits andfor other methods commonly used for the production of RNA. The use ofthe foamy virus reverse transcriptase-protease fusion protein with afunctionally inactivated protease provides a means for making largequantities of the fusion protein, such as by a recombinant bacterialhost cell, without the cleavage of the fusion protein typically seen inthese systems.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. An isolated nucleic acid encoding a polyprotein consisting of foamyvirus protease-reverse transcriptase having substantially reducedprotease activity as compared with wild-type protease-reversetranscriptase and a highly processive reverse transcriptase, wherein thefoamy virus protease-reverse transcriptase polyprotein comprises amutation in the aspartic acid residue in the protease catalytic site,and wherein the highly processive reverse transcriptase can produce anucleotide product of more than 600 basepairs in 10 minutes.
 2. Thenucleic acid of claim 1, wherein the nucleic acid encodes a modifiedsimian, cow, or human foamy virus protease-reverse transcriptase.
 3. Avector comprising a nucleic acid encoding a polyprotein consisting offoamy virus protease-reverse transcriptase having substantially reducedprotease activity as compared with wild-type protease-reversetranscriptase and a highly processive reverse transcriptase, wherein thefoamy virus protease-reverse transcriptase polyprotein comprises amutation in the aspartic acid residue in the protease catalytic site,and wherein the highly processive reverse transcriptase can produce anucleotide products of more than 600 basepairs in 10 minutes.
 4. Arecombinant host cell comprising a vector of claim
 3. 5. A kit for thepreparation of cDNA comprising an isolated foamy virus protease-reversetranscriptase polyprotein having a highly processive reversetranscriptase activity, and substantially reduced protease activity ascompared with wild-type protease-reverse transcriptase, wherein thefoamy virus protease-reverse transcriptase polyprotein comprises amutation in the aspartic acid residue in the protease active site, aprimer, an oligonucleotide template, deoxyribonucleotide bases, bufferssufficient for reverse transcription and a container and wherein thehighly processive reverse transcriptase can produce a nucleotide productof more than 600 basepairs in 10 minutes.